Presence Of P6 Research Articles

Multi-spectroscopic and molecular modeling studies of interaction between two different angiotensin I converting enzyme inhibitory peptides from gluten hydrolysate and human serum albumin

The present study was carried out to characterize Angiotensin-converting enzyme (ACE) inhibitory peptides which are released from the trypsin hydrolysate of wheat gluten protein. The binding of two inhibitory peptide (P4 and P6) to human serum albumin (HSA) under physiological conditions has been investigated by multi-spectroscopic in combination with molecular modeling techniques. Time-resolved and quenching fluorescence spectroscopies results revealed that the quenching of HSA fluorescence by P4 and P6 in the binary and ternary systems caused HSA-peptides complexes formation. The results indicated that both peptides quenched the fluorescence intensity of HSA through a static mechanism. The binding affinities and number of binding sites were obtained for the HSA-peptides complexes. The circular dichroism (CD) data revealed that the presence of both peptides increased the α-helix content of HSA and induced the remarkable folding of the polypeptide of the protein. Therefore, the CD data determined that the protein structure has been stabilized in the percent of ACE inhibitory peptides in binary and ternary systems. The binding distances between HSA and both peptides were estimated by the Forster theory, and it was revealed that nonradiative energy transfer from HSA to peptides occurred with a high probability. ITC experiments reveal that, in the absence and presence of P6, the dominant forces are electrostatic in binary and ternary systems. Furthermore, molecular modeling studies confirmed the experimental results. Molecular modeling investigation suggested that P4 bound to the site IA and IIA of HSA in binary and ternary systems, respectively. This study on the interaction of peptides with HSA should prove helpful for realizing the distribution and transportation of food compliments and drugs in vivo, elucidating the action mechanism and dynamics of food compliments and drugs at the molecular level. It should moreover be of great use for understanding the pharmacokinetic and pharmacodynamic mechanism of the food compliments and drugs.

Novel selective COX-1 inhibitors suppress neuroinflammatory mediators in LPS-stimulated N13 microglial cells

COX-1 plays a previously unrecognized part in the neuroinflammation. Genetic ablation or pharmacological inhibition of COX-1 activity attenuates the inflammatory response and neuronal loss. In this context, the effects of selective COX-1 inhibitors (P6, P10, SC-560, aspirin) and coxibs (celecoxib and etoricoxib) on LPS-stimulated microglial cell function (a worldwide accepted neuroinflammation model) were investigated, and the effects on COX-1/COX-2, cPGES mRNA and iNOS expression, PGE 2 and NO production and NF-κB activation by IκBα phosphorylation were evaluated. The total suppression of the expression of both COX-1 and COX-2 by their respective selective inhibitors occurred. NF-κB remained almost completely inactive in the presence of coxibs, as expected, and totally inactive in the presence of P6. P6 also markedly counteracted LPS enhancing cPGES mRNA expression and PGE 2 production. Since COX-1 is predominantly localized in microglia, its high selective inhibition rather than COX-2 (by coxibs) is more likely to reduce neuroinflammation and has been further investigated as a potential therapeutic approach and prevention in neurodegenerative diseases with a marked inflammatory component.

Nasal immunization with plasmid DNA encoding P6 protein and immunostimulatory complexes elicits nontypeable Haemophilus influenzae-specific long-term mucosal immune responses in the nasopharynx

Nasal vaccination is an effective therapeutic regimen for preventing upper respiratory infection, while DNA vaccines represent a new approach for controlling infectious diseases. Here, we examined the efficacy of nasally administered DNA vaccine on upper respiratory infections. A DNA plasmid encoding the P6 outer membrane protein of nontypeable Haemophilus influenzae (NTHi) was constructed. Mice were immunized 3 times intranasally with the DNA plasmid and Matrix-M, an immunostimulatory complex adjuvant. P6-specific immune responses were examined using purified P6 protein. Nasal-associated lymphoid tissue (NALT) CD4 + T cells were purified and incubated with feeder cells in the presence of P6, and the expression of cytokine mRNA was examined. In addition, NTHi challenges were performed and the level of NTHi was quantified in nasal washes. P6-specific nasal wash IgA and serum IgG were elevated following immunization with the DNA plasmid and Matrix-M. The number of specific IgA-producing cells increased in the nasal passages of the immunized mice. In addition to Th1 and Th2 cytokine expression, IL-17 was detected in P6-specific NALT CD4 + T cells. Moreover, DNA vaccination enhanced bacterial clearance. These findings suggest that a successful DNA vaccination protocol has been developed for inducing in vivo immune responses against NTHi. Nasal vaccination with P6 DNA vaccine and Matrix-M might be a new effective regimen for the induction of specific protective immunity in the upper respiratory tract.

Efficient DNA Packaging of Bacteriophage PRD1 Requires the Unique Vertex Protein P6

The assembly of bacteriophage PRD1 proceeds via formation of empty procapsids containing an internal lipid membrane, into which the linear double-stranded DNA genome is subsequently packaged. The packaging ATPase P9 and other putative packaging proteins have been shown to be located at a unique vertex of the PRD1 capsid. Here, we describe the isolation and characterization of a suppressor-sensitive PRD1 mutant deficient in the unique vertex protein P6. Protein P6 was found to be an essential part of the PRD1 packaging machinery; its absence leads to greatly reduced packaging efficiency. Lack of P6 was not found to affect particle assembly, because in the P6-deficient mutant infection, wild-type (wt) amounts of particles were produced, although most were empty. P6 was determined not to be a specificity factor, as the few filled particles seen in the P6-deficient infection contained only PRD1-specific DNA. The presence of P6 was not necessary for retention of DNA in the capsid once packaging had occurred, and P6-deficient DNA-containing particles were found to be stable and infectious, albeit not as infectious as wt PRD1 virions. A packaging model for bacteriophage PRD1, based on previous results and those obtained in this study, is presented.

Requirement of cauliflower mosaic virus open reading frame VI product for viral gene expression and multiplication in turnip protoplasts.

Cauliflower mosaic virus (CaMV) open reading frame (ORF) VI product (P6) has been shown to be the major constituent of viral inclusion body, to function as a post-transcriptional transactivator, and to be essential for infectivity on whole plants. Although these findings suggest that P6 has an important role in viral multiplication, it is unknown whether P6 is required for viral multiplication in a single cell. To address this question, we transfected turnip protoplasts with an ORF VI frame-shift (4 bp deletion) mutant (pCaFS6) of an infectious CaMV DNA clone (pCa122). The mutant was uninfectious. Co-transfection of plasmids expressing P6 complemented the mutant. Overexpression of P6 elevated the infection rate in co-transfection experiments with either pCa122 or pCaFS6. This would have been achieved by elevating the level of pregenomic 35S RNA, a putative polycistronic mRNA for ORFs I, II, III, IV and V, and by enhancing the accumulation of these five viral gene products. When CaMV ORFs I, II, III, IV and V were expressed from monocistronic constructs in which each of the ORFs was placed just downstream of the 35S promoter, the accumulation of ORF III, IV and V products depended on the co-expression of P6. The accumulation of ORF I and II products was not detected, even in the presence of P6. These results suggest that P6 is involved in the stabilization of other viral gene products as well as in the activation of viral gene expression, and thus, is a prerequisite for CaMV multiplication.

Multiple widely spaced elements determine the efficiency with which a distal cistron is expressed from the polycistronic pregenomic RNA of figwort mosaic caulimovirus.

The polycistronic expression mechanism of the plant pararetrovirus figwort mosaic caulimovirus (FMV) depends upon cis-acting elements present in its pregenomic RNA and a trans-acting protein (P6) which is expressed from a monocistronic subgenomic RNA. Using transient expression of FMV-derived polycistronic reporter constructs in Nicotiana edwardsonii cell suspension protoplasts, we further analyzed the cis-acting elements involved in polycistronic expression. A cis-acting element located within the first 74 nucleotides of the 7,954-nucleotide pregenomic RNA appears to be essential for P6 to transactivate expression of an internal cistron. Expression of this internal cistron, in the presence of P6, is greatly enhanced by the combined presence of two cis-acting elements located at the 3' end of the polycistronic RNA. Surprisingly, deletion of the most upstream of these two 3' cis-acting elements exposed a negative-acting element located internally on the polycistronic RNA, at the 3' end of open reading frame I. The action of both this negative-acting internal element and the positive-acting 3' elements is more pronounced when the large 5' untranslated leader region is present. This indicates that the 5' untranslated leader region is central to regulation of the FMV gene expression mechanism. Although a limited set of elements suffices to direct polycistronic expression in this eukaryotic system, a complex interplay between elements is involved in the spatial regulation of the genes present on the pregenomic RNA of FMV.

Functional domains of HIV-1 gag-polyprotein expressed in baculovirus-infected cells

Seven recombinants of AcNPV harboring various forms of complete or truncated gag gene from HIV-1 were constructed to determine which functional domains of the gag polyprotein are implicated in its self-assembly and cellular localization. The p6 car☐y-terminal portion of the p15 NC gag domain appeared to be dispensable for assembly, budding, and release of gag particles by insect cells. However, all the morphopoietic information was not entirely confined to the p9 NC domain, as N-myristylation could compensate for p15 NC deletion in gag assembly and the budding process. The two consensus karyophilic signals situated in the p17 MA gag domain were inefficient for targeting nonmyristylated forms of gag polyprotein to the nucleus when the p6 NC domain was deleted. In the presence of p6, or with a third, baculovirus-specific, karyophilic signal added at its N-terminus, gag particles relocated in the nucleus. These data suggested that p6 played a critical role in the conformation of gag polyprotein.